Studies on an Ad2 soluble DNA replication complex. We will further investigate the soluble DNA replication complex (SC) isolated from the nuclei of Ad2 infected KB cells. The DNA product will be characterized with regard to (1) the segments and strands of the viral genome that are synthesized in vitro, (2) whether DNA termini are completely finished in vitro, and (3) whether the 5'-terminal covalently linked protein is attached to in vitro completed DNA. The enzyme activities (DNA polymerase alpha and gamma, RNA polymerase II, DNA ligase, RNase, and DNase) and viral induced early proteins that we find associated with SC will be further studied to establish possible function by further purification, inhibition studies, and reconstitution studies. Methylation of Ad2 early mRNA. Ad2 early mRNA is transcribed and processed by host cell enzymes, and thus provides a model for the study of gene expression in eukaryotic cells. We have described the methylation pattern of Ad2 early mRNA, and have provided evidence for the existence of at least 7 different 5'-methylated cap sequences. We propose to select individual early mRNA molecules by preparative hybridization to restriction fragments in order to establish the methylation pattern of each early viral mRNA coded by different regions along the viral genome. This will help establish the role of template DNA and host mechanisms in determining the methylated cap sequence.